A large fraction of beer is produced on the basis of barley (Hordeum vulgare, L.), which is a monocotyledonous crop plant grown in many parts of the world. It is propagated not only due to its economic importance as a source of industrial products, such as beer, but also as a source of animal feed.
Methods are unfortunately not available for preparing transgenic barley plants that completely lack expression of a given gene and corresponding protein. In general for barley, application of antisense techniques may be utilized to generate transgenic plants that still express some of the protein (see, for example, Robbins et al., 1998; Stahl et al., 2004; Hansen et al., 2007). Also, effective methods have not been developed for preparing specific mutations using chimeric RNA/DNA or site-directed mutagenesis in barley plants. In fact, not a single example of successful oligonucleotide-directed gene targeting in barley has been published. Iida and Terada (2005) noted that oligonucleotide-directed gene targeting has been pursued in maize, tobacco and rice, but not in barley—and in all cases with the ALS gene as a target. Part of the research conclusion was that it remains to be seen whether the strategy with appropriate modifications can be applicable to genes other than those directly selectable, such as the ALS genes. Targeted mutagenesis using zinc-finger nucleases represents another tool that could be used in the future to investigate basic plant biology or to modify crop plants (Durai et al., 2005; Tzfira and White, 2005; Kumar et al., 2006). Also in this case, mutagenesis has not been pursued or successfully applied in barley.
Barley mutants, however, may be prepared by random mutagenesis using chemical treatment or irradiation, such as by treatment with sodium azide (NaN3; FIG. 1). An example is barley kernels mutagenized with NaN3, and screened for high levels of free phosphate, with the aim to identify low-phytate mutants (Rasmussen and Hatzack, 1998); a total of 10 mutants out of 2,000 screened kernels were identified. However, identification of a particular mutant after NaN3 treatment requires an effective screening method and is far from always successful.
In 1970, the molecule conferring the cardboard-like flavor in beer was isolated and identified as T2N, a volatile C9 alkenal (Jamieson and Gheluwe, 1970). Since the taste-threshold level for T2N in humans is extremely low, previously determined to be around 0.7 nM or 0.1 ppb (Meilgaard, 1975), products with even minute levels of the aldehyde are regarded as being aged due to the off-flavor taste of the product. However, the T2N level is generally very low in fresh beer (Lermusieau et al., 1999), so it has been speculated that during storage, free T2N may be liberated from T2N adducts (Nyborg et al., 1999). This notion was supported by a subsequent observation that the T2N potential in wort correlates with formation of T2N after product storage (Kuroda et al., 2005).
The barley kernel contains three LOX enzymes known as LOX-1, LOX-2, and LOX-3 (van Mechelen et al., 1999). LOX-1 catalyzes the formation of 9-hydroperoxy octadecadienoic acid (9-HPODE; see FIG. 2 for a partial overview of LOX pathway)—a precursor of both T2N and trihydroxy octadecenoic acids (abbreviated THAs)—from linoleic acid. LOX-2 mainly catalyzes the conversion of linoleic acid to 13-HPODE, which is further metabolized to hexanal, a C6 aldehyde with a ˜0.4-ppm-high taste threshold (Meilgaard, supra). LOX-3 action is probably not of relevance with respect to the instant application for two reasons: the expression level of the corresponding gene in barley kernels is very low, and the product specificity of LOX-3 remains elusive.
In support of the aforementioned data, several reports have noted that T2N is produced via a biochemical pathway involving conversion of linoleic acid to 9-HPODE, initially catalyzed by LOX-1, and then cleavage of 9-HPODE through 9-hydroperoxide lyase action (see, for example, Kuroda et al., 2003, 2005; Noodermeer et al., 2001).
There appears to be no correlation between the overall LOX activity in malt and the wort nonenal potential. However, there have been speculations about a significant correlation between LOX-1 activity and wort T2N potential, primarily because LOX-2 activity was considered inferior with respect to formation of the T2N potential in wort (Kuroda et al., 2005).
In FIG. 2 is, as mentioned above, shown a part of the LOX pathway, here focusing on biochemical reactions from linoleic acid to T2N. The major activity of the LOX-1 enzyme concerns the conversion of linoleic acid to 9-HPODE, which is an upstream metabolite of the biochemical pathway leading to formation of T2N. In contrast, the major activity of LOX-2 relates to the conversion of linoleic acid into 13-HPODE, which is separate from the aforementioned biochemical pathway to T2N. It is notable that LOX-1 and LOX-2 enzymes may utilize linolenic acid as substrate, but this activity is outside the scope of the instant application as the corresponding pathways do not lead to T2N formation.
LOX-1 has been thought to contribute with the major LOX activity in malt (see, for example, Kuroda et al., 2003).
Several different barley plants have been developed, which are characterized by reductions in, or lack of, LOX-1 activity. For example, barley kernels and barley plants having a low LOX-1 activity were disclosed in PCT application WO 02/053721 to Douma, A. C. et al. And in WO 2005/087934 to Breddam, K. et al., attention was on two different barley mutants deficient in LOX-1 activity—a splice mutant and a mutant with a premature translational stop codon. These were identified following propagation and screening of mutated plants, as illustrated in FIG. 1. While the above-mentioned mutants were identified by screening NaN3-mutagenized barley, Hirota, N. et al. described in EP 1609866 a barley plant with no LOX-1 activity, which was identified by screening a collection of barley landraces.
Several examples on mutated plants that synthesize low levels of LOX are known. However, no barley plant deficient in several lipoxygenase activities, for example no barley plant deficient in activities of both LOX-1 and LOX-2 has been described. Methods to enable genetic manipulation of plants are frequently specific to a specific kind of plant, and thus—despite that few rice plants, soy plants, or Arabidopsis plants are known to comprise low levels of LOX—the methods for preparing such plants cannot be used in the generation of barley plants with low, or no, LOX activity. In addition, LOX mutants of one plant species may have different properties in comparison with LOX mutants of another plant species.